In this study, we used time-locked optogenetic inhibition to silence VTA projections towards the NAcbS during 5-CSRTT performance in rats. We found that reducing activity of these projections resulted in exaggerated premature responding. This effect was only present when inhibition took place near the end of the ITI, highlighting the temporal importance of this projection. Furthermore, these effects were selective for premature responding since they did not affect attentional or motivational performance. Demonstrating that inhibition of VTA inputs to the NAcbS, in a timing-specific manner, increases impulsive action.
Earlier studies have provided substantial evidence for the involvement of DAergic transmission in the NAcbS in impulsivity in the 5-CSRTT (Besson et al, 2010; Dalley and Robbins, 2017; Economidou et al, 2012; Moreno et al, 2013; Murphy et al, 2008; Pezze et al, 2007). Pharmacological studies have demonstrated that premature responding is associated with high levels of DA in this region (Dalley and Robbins, 2017; Diergaarde et al, 2008; Robbins, 2018; Murphy et al, 2008; Jupp et al, 2013), which is in line with the exacerbating effect of VTA-NAcbS activation on premature responses we found previously (Flores-Dourojeanni et al. 2021).
It is important to emphasize that halorhodopsin has been previously known to induce a post-inhibitory excitation of neurons upon termination of laser stimulation (Mattingly et al. 2018). This is thought to be a result of a lowered threshold of firing caused by alterations in the ionic gradient. However, the effects observed on premature responses occurred within the period of inhibition (Mattingly et al. 2018). Since the post-inhibitory effects of halorhodopsin occur after inhibition (after the laser has been turned off) we can safely discount this as an alternative explanation of our results. Nevertheless, the relationship between DA and impulsivity remains unclear. As demonstrated by our previous studies wherein stimulating VTA DA neurons did not increase premature responding (Flores-Dourojeanni et al. 2021; Boekhoudt et al. 2017). The complexity of DA’s role in impulse control may be a result of the different DA receptor subtypes within the NAcb. The principal cell types in the NAcb are GABAergic medium spiny neurons (MSNs), which can be categorized into two functionally distinct populations based on their expression of either the D1 or D2/3-type DA receptor. Pharmacological studies have found differential effects of DA D1 and D2/3 receptor inactivation on 5-CSRTT performance. Local infusion of a DA D1 receptor antagonist into the NAcbS of rats reduced impulsivity on the 5-CSRTT (Pattij and Vanderschuren 2008), whereas infusions of a DA D2/3 receptor antagonist increased impulsive premature responding (Besson et al. 2010). Furthermore, trait-like impulsivity in rats is linked to decreased DA D2/3 receptor availability in the NAcbS (Besson et al. 2010; Dalley and Robbins 2017; Jupp et al. 2013). Oral administration of methylphenidate has been found to alleviate this exaggerated premature responding in highly impulsive rats, while also upregulating DA D2/3 receptor availability in the ventral striatum (Caprioli et al. 2015). This suggest that optogenetic inhibition of NAcbS projecting VTA neurons causes an increase in premature responding by decreasing the activity of DA D2/3 receptors. Moreover, it is possible that the increase in premature responding previously observed during optogenetic stimulation may be driven by changes in D1 receptor signalling. Although this remains to be tested. Put together, our findings imply that impulse control in the 5-CSRTT requires a balance of DA levels in the NAcbS during the critical time-point preceding the cue. Disrupting this balance by changing VTA to NAcbS signalling induces exaggerated impulsive premature responding.
Previously we found that chemogenetic activation of VTA DA neurons increases the number of omissions (Boekhoudt et al. 2017). Similarly, we also showed that activating VTA efferents towards the NAcbS or the NAcbC provide the same effect (Flores-Dourojeanni et al. 2021). In this study, however, we show that inhibition of VTA-NAcbS projections does not alter the number of omissions. To date, the role of DA transmission on errors of omission in the 5-CSRTT is not entirely clear. Region specific studies that have investigated the role of D1 and D2/3 DA receptors in the NAcbS have found that infusion of both D1 and D2/3 antagonists increase the number of omissions in the 5-CSRTT (Pattij et al. 2007; Besson et al. 2010; Pezze et al. 2007). In contrast, inactivation of the NAcbS via infusions of the GABA agonist muscimol does not alter the number of omissions (Feja et al. 2014). The lack of attentional effects in this experiment may indicate that impulse control is more sensitive to fluctuations in NAcb DA than attention. After all, optogenetic silencing is partial (not all projecting neurons will express NpHr) and time-locked (restricted to a portion of the ITI). These aspects can allow for enough neuronal signalling to pass through and enable proper attentional behaviour. Alternatively, VTA projections to the NAcbC may compensate for the inhibition of NAcbS efferents since both regions have been shown to play a role in attentive behaviour.
Our optogenetic manipulations targeted VTA neurons that innervate the NAcbS. Although this projection also comprises non-DA neurons (Morales and Margolis 2017; Yamaguchi et al. 2011), it is well-established that over 80% of the VTA projections to the NAcb emerge from DA neurons (Swanson 1982). Consistent, previous studies in our lab, using the same two-viral approach in this pathway, have found a similar proportion of infected VTA-NAcb neurons to be DAergic (Boender et al. 2014; Verharen et al. 2018). Moreover, using in-vivo microdialysis, we have shown increased baseline levels of DA and its metabolites in the NAcb after chemogenetic activation of this projection (Verharen et al. 2018). Nevertheless, a contribution from GABAergic and glutamatergic neurons may still play an important role in mediating the observed behaviours. For instance, muscimol infusions into the NAcbS has been shown to increase premature responses in the 5-CSRTT (Feja et al. 2014). However, GABA infusions in that study also greatly increased all response latencies whereas we found an increase in premature responses in the absence of any latency changes. This may indicate that our manipulation is driven by a different mechanism. Also, inhibition of GABAergic neurons in the VTA would cause a disinhibition of neurons in the NAcbS which would contrast the effects of a GABA agonist in the NAcbS. However, since we’ve demonstrated that both inhibition and stimulation of VTA-NAcbS efferents disrupts impulse control, the idea that a fine balance of stimulation and inhibition is necessary to maintain proper impulse control, may still hold true for either DA or GABAergic transmission. Concerning glutamatergic neurons, optogenetic inhibition of VTA Vglut2 + neurons projecting to the NAcb have been shown to prevent foot-shock driven increased immobility in a forced swim test (Qi et al. 2016). These results suggest glutamatergic neurons in the VTA may play a role in behavioural inhibition within the NAcb.
Altogether this study complements our previous findings showcasing the importance of VTA projections towards the NAcbS. By inhibiting cells with time-locked precision during the last seconds of the ITI of the 5-CSRTT we were able to increase premature responding without altering any other parameters. We show, together with our previous findings, that a fine balance of activity emerging from the VTA towards the NAcbS is essential for maintaining proper inhibitory control.